Intercepting IRE1 kinase‐FMRP signaling prevents atherosclerosis progression

Abstract Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA‐binding protein (RBP) that controls translation of select mRNAs. We discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP‐bound mRNAs. We show ER stress‐induced activation of Inositol requiring enzyme‐1 (IRE1), an ER‐resident stress‐sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress‐induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis.

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EMBO Molecular Medicine has a "scooping protection" policy, whereby similar findings that are published by others during review or revision are not a criterion for rejection. Should you decide to submit a revised version, I do ask that you get in touch after three months if you have not completed it, to update us on the status. In this manuscript, Yildirim and colleagues identify a novel function of FMRP protein in macrophages, which is well-known for its crucial in normal brain development. The authors present clear data showing that the ER stress sensor IRE-1 phosphorylates FMRP protein, which results in suppression of macrophage cholesterol efflux and apoptotic cell clearance. As FMRP is an RNA binding protein, the authors show that FMRP phosphorylation enhances the translational suppression of major cholesterol transporters and efferocytosis receptors, including ABCA1, ABCG1, MERTK and LRP1. Furthermore, using established mouse models of atherosclerosis, the authors show that FMRP deficiency or systemic IRE-1 kinase inhibition lowers en face aortic lesion development and inhibits necrotic core formation in the aortic roots. The results in this study are of good quality and in the interest of the field as ER stress contributes many aspects of obesity-induced metabolic perturbations, including atherosclerosis. The authors present strong data to support their proposed mechanism, and some aspects of the manuscript could be improved by considering some suggestions listed below.
1-Previous work from the same group has shown that inhibiting IRE1's both kinase and endoribonuclease activities in macrophages suppresses hyperlipidemia-induced IL-1β and IL-18 production, which contributes to the beneficial effect of IRE-1 inhibition in lowering atherosclerosis formation in ApoE-/-mice. Have the authors examined the levels of IL-1β and/or IL-18 in FMRP-/-or AMG-18 treated macrophages? 2-The authors show lower TUNEL-positivity and plaque necrosis in both FMRP-deficient and AMG-18 treated ApoE-/-mice. As lower TUNEL positivity in lesions reflects both enhanced efferocytosis and apoptosis suppression, have the authors attempted to measure whether ER stress-induced macrophage apoptosis is decreased in FMRP-/-and/or AMG-18-treated macrophages ex vivo? 3-Can the authors show or discuss whether FMRP is the only mediator downstream of IRE1 in regulating macrophage cholesterol efflux and/or apoptotic cell clearance?
Referee #3 (Comments on Novelty/Model System for Author): The current study investigates the role of FMRP in cholesterol efflux and efferocytosis both in cell culture experiments and in vivo models in mice, including an atherosclerosis model. This is largely an unexplored area, and the investigators use a variety of experimental approaches to support their conclusions, which strengthens the study considerably.
Referee #3 (Remarks for Author): The current study investigates the role of FMRP in cholesterol efflux, efferocytosis both in cell culture experiments and in vivo models in mice. The results demonstrate that ER stress-induced activation of IRE1 leads to FMRP phosphorylation and suppression of macrophage cholesterol efflux and efferocytosis. Further, the study demonstrates that FMRP-deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis. The final experiments examine FMRP-deficiency (global) as well as in myeloid cells as well as IRE1 inhibition on the progression of atherosclerosis in various mouse models.
Overall, this is an important study that is carefully done with numerous experiments to confirm that largely confirm the conclusions reached by the authors. A few items need to be addressed that would strengthen the paper: 1. There is some concern about certain aspects of data analysis. For example, it's not clear how the immunoblot data which is prevalent throughout the study were analyzed. In almost all cases it appears the control was assigned a value of 1.0. As an example, the data in S1F, H, and I, the IRE+/+ results (pFMRP/FMRP) are always set to 1.0. Obviously, there is variation in these samples, which would impact the significance. The same problem is true for the data in S3C and S3D.
2. It does not look like very many residential macrophages in the control group (DMSO) are oil-red O positive in Fig 2B. 3. In Figs 2C,D the authors refer to "cholesterol accumulation" and Fig 2E refer to "reduced foam cell formation". The experiments are measuring %dil-acLDL internalized; I would recommend that they refer to the actual event they are measuring to be accurate in their descriptions. Fig 2C,D, on page 6, line 217 the authors state that "We reasoned that this observation could be related to an increase in cholesterol export (RCT; due to increased translation of cholesterol exporters) from Fmr1-/-macrophages." While this appears to be the case, the measured data show decreased levels of diI-ac-LDL in cells. This could also be impacted by reduction in receptors involved in uptake as well. The authors might consider mentioning this possibility.

Based on the results of experiments in
5. Fig 4D, differences between EV WT or STSA are extremely small. Once again, there is a question about data analysis. Were the data normalized to EV (all EV values are 1.0). Variations in EV would certainly alter statistical analysis.

Referee #2 (Comments on Novelty/Model System for Author):
The authors present a clear and systematic rationalization and evaluation of the role of FMRP in macrophages and in atherosclerosis development.

Referee #2 (Remarks for Author):
In this manuscript, Yildirim and colleagues identify a novel function of FMRP protein in macrophages, which is well-known for its crucial in normal brain development. The authors present clear data showing that the ER stress sensor IRE-1 phosphorylates FMRP protein, which results in suppression of macrophage cholesterol efflux and apoptotic cell clearance. As FMRP is an RNA binding protein, the authors show that FMRP phosphorylation enhances the translational suppression of major cholesterol transporters and efferocytosis receptors, including ABCA1, ABCG1, MERTK and LRP1. Furthermore, using established mouse models of atherosclerosis, the authors show that FMRP deficiency or systemic IRE-1 kinase inhibition lowers en face aortic lesion development and inhibits necrotic core formation in the aortic roots.
The results in this study are of good quality and in the interest of the field as ER stress contributes many aspects of obesity-induced metabolic perturbations, including atherosclerosis. The authors present strong data to support their proposed mechanism, and some aspects of the manuscript could be improved by considering some suggestions listed below.

Response:
We thank the reviewer for recognizing the novelty of our findings and the good quality of our data. We appreciate these encouraging remarks and the reviewer's constructive feedback, which we aspired to build upon in our revised manuscript.
Question 1: Previous work from the same group has shown that inhibiting IRE1's both kinase and endoribonuclease activities in macrophages suppresses hyperlipidemia-induced IL-1β and IL-18 production, which contributes to the beneficial effect of IRE-1 inhibition in lowering atherosclerosis formation in ApoE-/-mice. Have the authors examined the levels of IL-1β and/or IL-18 in FMRP-/-or AMG-18 treated macrophages?
Response 1: We thank the reviewer for pointing out our group's prior published findings that described IRE1's endoribonuclease activity inhibition contributes to both hyperlipidemia-induced inflammation and atherosclerosis formation. In this study, for the first time, we attempted to investigate IRE1's kinase activity and novel substrate, FMRP's role in atherosclerosis. Indeed, hypercholesterolemia and inflammation are 3rd Jan 2022 1st Authors' Response to Reviewers intertwined and both drive atherogenesis. As suggested by the reviewer, we investigated the impact of FMRP deficiency on lipid-induced inflammation, specifically IL-1b, in macrophages. As seen below (A and B), FMRP loss of function (in both Fmr1 knock down that were transfected with Fmr1-specific siRNA and FMRP knock out bone marrow-derived mouse macrophages (BMDM)), abrogated lipid-induced m-IL-1b secretion. However, knocking out FMRP had no impact on Fmr1 mRNA associated with the polysomes or in the whole cell lysates (C and D). FMRP loss of function also did not appear to alter lipid-induced inflammasome activation as assessed by active caspase-1 in the conditioned medium of these macrophages (A and B). Supernatants were analyzed by western blotting using specific antibody for IL-1β and caspase-1 and protein lysates were analyzed by western blotting using specific antibodies for FMRP, pIRE1, IRE1 and b-Actin.
As suggested by the reviewer, we also treated bone marrow derived mouse macrophages (BMDM) with AMG-18, which reduced lipid-induced mature IL-1b (m-IL-1b) secretion (seen in E).   ., 2017). FMRP suppression also reduces mIL-1β secreted from macrophages but without altering inflammasome activation (Appendix Fig S6A-S6F). Knocking out FMRP from macrophages has no effect on IL-1β mRNA levels in the whole cell lysate or in the polysomes, suggesting against transcriptional or translational control over these cytokines' production (Appendix Fig S6G and S6H). As expected, inhibition of IRE1 kinase also reduces mIL-1β (Appendix Fig S6I-S6K). Intriguingly, IL-1β can be secreted through Response 2: We share the concern for this possibility with the reviewer and as suggested by the reviewer, we assessed apoptosis in macrophages. As seen below, we observed that both FMRP genetic deletion and IRE1 kinase inhibition did not alter apoptosis (as measured by PI positive cells using flow cytometry). This finding supports the notion that enhanced efferocytosis is the primary consequence of inhibiting IRE1-FMRP pathway. The below graph (as Appendix Figure S4I) and the relevant results discussion was added to our revised manuscript: "We further investigated whether apoptosis is altered in Fmr1 -/and AMG-18 treated macrophages. There was no significant change between the groups (Appendix Fig 4I), supporting the notion that the primary consequence of inhibiting IRE1-FMRP signaling is efficient clearance of apoptotic cells through increasing efferocytosis capacity."

Referee #3 (Comments on Novelty/Model System for Author):
The current study investigates the role of FMRP in cholesterol efflux and efferocytosis both in cell culture experiments and in vivo models in mice, including an atherosclerosis model. This is largely an unexplored area, and the investigators use a variety of experimental approaches to support their conclusions, which strengthens the study considerably.

Referee #3 (Remarks for Author):
The current study investigates the role of FMRP in cholesterol efflux, efferocytosis both in cell culture experiments and in vivo models in mice. The results demonstrate that ER stress-induced activation of IRE1 leads to FMRP phosphorylation and suppression of macrophage cholesterol efflux and efferocytosis.
Further, the study demonstrates that FMRP-deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis. The final experiments examine FMRP-deficiency (global) as well as in myeloid cells as well as IRE1 inhibition on the progression of atherosclerosis in various mouse models.
Overall, this is an important study that is carefully done with numerous experiments to confirm that largely confirm the conclusions reached by the authors. A few items need to be addressed that would strengthen the paper:

Response:
We thank the reviewer for his/her enthusiastic evaluation of our study's novel findings and for the reviewer's constructive feedback that encouraged us to improve our manuscript.

Question 1:
There is some concern about certain aspects of data analysis. For example, it's not clear how the immunoblot data which is prevalent throughout the study were analyzed. In almost all cases it appears the control was assigned a value of 1.0. As an example, the data in S1F, H, and I, the IRE+/+ results (pFMRP/FMRP) are always set to 1.0. Obviously, there is variation in these samples, which would impact the significance. The same problem is true for the data in S3C and S3D.
Response 1: We thank the reviewer for giving us an opportunity to clarify how the western blot data analysis was performed. In order to assess the reviewer's concern, we updated our quantifications (specifically for figures with quantified blots such as ( Figure 1C, 1D Below is the detailed explanation for fold change calculation and a sample calculation for Figure 1D. We used a standard approach utilized by many labs to analyze the changes on FMRP phosphorylation across multiple experimental set ups (plated, treated, lysed on different days) or gels/blots run/developed on different days. We first determined the ratio for P-FMRP/ FMRP band intensity for all controls and samples. We set the value for the control with the lowest ratio as "1.0" and determined the fold change for the other controls and treatment samples on the same gel/blot. If there were additional experimental sets run on different gels, we did the same calculation for each gel/blot. After this we combined all the experimental sets by first determining the average of the control samples in all sets/gels/blots and assigning this average with the value of 1.0. Then, we took the average (of the fold change) for a certain treatment group determined the average mean value for the fold change.
One exception to this calculation was Figure 1C (that was quantified in Appendix Figure S1A

Response 3:
We agree with the reviewer and accordingly, revised our description of the outcome by only referring to the outcome of these experiments in Figures 2C, 2D, and 2E as these treatments "reduced % dil-acLDL internalized." Question 4: Based on the results of experiments in Fig 2C,D, on page 6, line 217 the authors state that "We reasoned that this observation could be related to an increase in cholesterol export (RCT; due to increased translation of cholesterol exporters) from Fmr1-/-macrophages." While this appears to be the case, the measured data show decreased levels of diI-ac-LDL in cells. This could also be impacted by reduction in receptors involved in uptake as well. The authors might consider mentioning this possibility.

Response 4:
We thank the reviewer for this great feedback. As seen in Appendix Figure S2A, neither FMRP knockdown nor IRE1 kinase inhibition alters cholesterol uptake in macrophages. This result supports the conclusions that decreased foam cell formation is due to increased cholesterol export in both FMRP-deficient and IRE1 kinase-inhibited macrophages. We added the below graph to Fig. S2 and explained in the revised manuscript that "Reduced foam cell formation could be explained with less cholesterol uptake, however, neither FMRP knock down nor IRE1 kinase inhibition altered cholesterol uptake in macrophages (Appendix Fig S2B). We reasoned that this observation is most likely related to an increase in cholesterol export (RCT; due to increased translation of cholesterol exporters) from Fmr1-/macrophages. Indeed, FMRP deficiency led to an increase in cholesterol efflux coupled to its loading onto the cholesterol carriers, apolipoprotein-A1 (APOA1) and HDL (Fig 2F), and, likewise, the IRE1 kinase inhibitor enhanced cholesterol efflux (Fig 2G). Thus, ER stress-associated reduction in cholesterol efflux is dependent on both IRE1 kinase activity and FMRP." in our revised manuscript. Response 5: Please, see our response to question 1 for a detailed explanation on our data analysis from Western blots. As advised by the reviewer, we updated our calculations for fold change of FMRP target proteins, which occurs in the range of 25-35% when WT_FMRP is compared to STSA_FMRP mutant as seen below (and in the revised Fig.4D and Appendix Fig.S3D):
Response 1: We revised the description of the experiment as "Endogenous FMRP and IRE1, migrated faster on SDS-PAGE than epitope tagged EGFP-FMRP and FLAG-IRE1, respectively". Question 2: Page 5, line 207 "Next, we fed Apoe-/-mice with a WD (12 weeks) and injected them daily with the IRE1 kinase inhibitor" Not sure why ? is present Response 2: We thank the reviewer for pointing out this typing mistake. We deleted the question mark (?) in our revised manuscript. Dear Dr. Erbay, Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed reports from the referees. As you will see, they are fully supportive of publication, and I am therefore pleased to inform you that we will be able to accept your manuscript once the following editorial points will be addressed: 1/ Main manuscript text: -Please address the queries from our data editors in track changes mode in the main manuscript file labelled 'Data edited MS file'. Please use this file for any further modification (in track changes mode).
-We can accommodate a maximum of 5 keywords, please adjust accordingly. 4/ Thank you for providing a synopsis. I slightly shortened the text to fit our style and format, please let me know if you agree with the following: Targeting IRE1 function and substrate(s) provides a novel therapeutic approach to atherosclerosis. We found a key role for IRE1-mediated FMRP phosphorylation that suppresses the expression of cholesterol transporters and efferocytosis receptors in macrophages and promotes atherosclerosis progression.
• FMRP is a novel IRE1 kinase substrate.
• Ablation of IRE1 kinase activity or suppression of FMRP expression enhances efferocytosis and cholesterol transport in vitro and in vivo.
• IRE1 kinase inhibition by a small molecule inhibitor or genetic deletion of FMRP in macrophages alleviates atherosclerotic plaque formation.
Please upload your synopsis image as an independent tiff, jpeg or PNG file 550 px wide x 300-600 px high. The text should remain legible. 5/ As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329), EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts. This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. Let us know whether you agree with the publication of the RPF and as here, if you want to remove or not any figures from it prior to publication. Please note that the Authors checklist will be published at the end of the RPF. The system will prompt you to fill in your funding and payment information. This will allow Wiley to send you a quote for the article processing charge (APC) in case of acceptance. This quote takes into account any reduction or fee waivers that you may be eligible for. Authors do not need to pay any fees before their manuscript is accepted and transferred to our publisher. Dear Dr. Erbay, Thank you for submitting your revised files. I am pleased to inform you that your manuscript is accepted for publication and is now being sent to our publisher to be included in the next available issue of EMBO Molecular Medicine.
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